The DnaK (Hsp70), DnaJ, and GrpE heat shock proteins of Escherichia coli constitute a cellular chaperone system for protein folding. Substrate interactions are controlled by the ATPase activity of DnaK which itself is regulated by the nucleotide exchange factor GrpE. To understand the structure-function relationship of this chaperone system, the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes were analyzed by gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and native gel electrophoresis. GrpE formed dimers in solution. DnaK formed monomers, dimers, and higher mole mass oligomers, the equilibrium between these forms being dependent on the DnaK concentration. The behavior of DnaK and GrpE in gel filtration and dynamic light scattering suggested elongated shapes of both molecules. In the absence of added nucleotides, DnaK and GrpE formed stable complexes containing one molecule of DnaK and two molecules of GrpE. A 44-kDa N-terminal ATPase fragment of DnaK also formed complexes with GrpE with the same 1:2 stoichiometry. DnaK-GrpE complex formation was unaffected by elimination of DnaK-bound nucleotides or addition of saturating concentrations of a DnaK peptide substrate. These findings allow the correlation of DnaK-GrpE interactions with a role for GrpE in the functional cycle of the DnaK chaperone system.

The specific attachment of bathophenanthroline‐ruthenium(II) complexes as non‐radioactive label molecules to synthetically 5′‐NH2‐modified oligonucleotides is described. After excitation by light pulses, the fluorescence of these labels can be measured by a time‐resolved mode woth high sensitivity. No quenching takes place due to coupling of the Ru complexes to the DNA. Ru‐complex‐labelled oligonucleotides still hybridize specifically to complementary DNA sequences, and no quenching is observed in the course of the hybridization process.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.

Nerve growth factor (NGF) binds to two neurotrophin receptors: p75NTR and p140Trk (TrkA). Both receptors dimerize in response to NGF binding. TrkA homodimers and heteromers of TrkA and p75NTR promote cell survival whereas homodimers of p75NTR mediate apoptosis upon binding of NGF. The interaction between receptor and NGF can be inhibited either on the level of the ligand by altering NGF conformation so that NGF is no longer recognized by the receptor or on the level of the receptor by blocking the binding site of p75NTR or TrkA. The effect of altering NGF conformation on NGF signaling was investigated in two neuron‐like cell lines: in human SK‐N‐MC cells that express only p75NTR and in rat PC12 cells that express both p75NTR and TrkA. In the present study we demonstrate that Ro 08‐2750 binds to the NGF dimer thereby probably inducing a change in its conformation such that NGF cannot bind to p75NTR anymore. In SK‐N‐MC cells this leads to inhibition of NGF‐induced programmed cell death. In PC12 cells enhanced signaling through TrkA was observed. J. Neurosci. Res. 61:263–272, 2000. © 2000 Wiley‐Liss, Inc.

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